Lab-on-chips, usually operating as microfluidic devices, are constructs that facilitate a variety of assays of biological liquid samples, e.g. blood, blood plasma or urine. Nowadays, microfluidic systems are becoming of increased importance as they require reduced sample usage to be investigated due to their miniaturization.
One of the assays that have been applied to microfluidic systems includes agglutination tests of blood. Typically, the measurement involves a reaction of the blood cells or the whole blood with the substance acting as a reagent, i.e. an agglutination agent (antibody), within channel(s) (such as microfluidic channel(s)), wherein the reagent causes a change in certain physical properties of the investigated blood, so that the agglutination may be determined by measuring the change. Typically, blood typing is performed with the use of blood fractions, i.e. after centrifugation. Blood cells isolated from the whole blood are then tested in contact with monoclonal reagents containing antibodies, and isolated plasma is tested with standardized red blood cells with known antigens on their surface.
Another category of serological tests are immunoassays, such as PCR and ELISA.
The most widely known methods for determining agglutination involve visual assessment of clumping of blood cells in the sample, which can be conducted either in a large scale, e.g. on glass plates and in test tubes, or in miniaturized scale with microfluidic systems.
For example, the U.S. Pat. No. 8,318,439 describes a microfluidic apparatus for blood typing by visual determination of appearance of the aggregates resulting in agglutination process. The device comprises an agglutination reaction channel provided with blood supply, an agglutination reagent supply, a throat and an optical window wherein the positive reaction may be optically assessed by appearance of “clumps” which are visible in the window. Further, the selected dimensions of the throat influence on the Reynolds number, and therefore potentiate the agglutination reaction. Nevertheless, the visual method may cause generation of errors due to the imperfections of human eye.
In addition, various other documents describe devices and methods for determining agglutination of the biological liquid samples by comparison of change in blood viscosity.
For instance, a PCT patent application WO2012164263 describes a microfluidic system and an assay method, which involve measuring a variation in viscosity of a sample. The microfluidic device consists of two parallel channels intersected at their inlets and outlets. The method involves adding blood sample into the microfluidic channels followed by providing a chase buffer into the channel. The liquid flows along the first and the second channel, wherein the agglutination agent is provided in the first, but not in the second channel. As the agglutination slows the flow rate of the blood sample, only non-agglutinated fluid may fall out of the channels arrangement, and thus block the agglutinated sample in the first channel. The distance travelled by the agglutinated liquid in the first channel is indicative of the degree of agglutination.
A similar method and device are described in a US patent application US20090317793. The device comprises a reference channel and a test channel in which an agglutination agent is provided. The channels are intersected at their both ends and provided with merging regions arranged such that the reference liquid flowing from the upper reference channel section into the merging region blocks a test liquid flow in the upper test channel. The agglutination is determined visually by observation of the position of flow of front of the sample liquid in the test channel.
Another diagnostic assay method is described in a PCT patent application WO2011035385. The method involves agglutination of a sample, followed by its deposition on a porous analytical substrate, on which the sample wicks. The sample that agglutinated upon contact with the specific antibodies separates/elutes upon contact with the substrate, while the blood sample upon contact with non-specific antibody does not separate/elute. The elution velocity and the extent of sample separation on the substrate indicate the coagulation degree.
A US patent application US20030224457 describes a method for determining the presence of antibodies in a blood sample by reverse typing on an optical bio-disc provided with a microfluidic channel; the method involves applying a blood sample into the microfluidic channel followed by spinning a disc which effects on movement of a blood serum through the microfluidic channel; next, adding to the serum a known ABO blood group and spinning the optical bio-disc, then incubating the mixture within the microfluidic channel. The agglutination is measured by scanning the mixture with an incident beam of electromagnetic radiation. Data obtained by irradiation determines the presence of agglutinated cells.
Thus, there are numerous ways to determine agglutination with microfluidic devices. However, in certain circumstances, the agglutination reaction may cause a cessation of flow and thus, the sample may be blocked, which may further lead to erroneous estimation of the agglutination assay income. In addition, it may cause difficulties in cleaning of the microfluidic channels. Moreover, when using a smaller sample, i.e. a sample having a size of a droplet or a droplet, or in case when agglutination reaction degree is substantially meager, typically, the agglutination phenomenon might not be assessed correctly.
A polish patent application PL396494 describes a device and a method for conducting agglutination test with a microfluidic system using a droplet size blood sample. The microfluidic system is provided with an antibody carrier droplet (such as monoclonal reagents) supply, an antigen carrier droplet (e.g. red blood cells) supply and a microfluidic channel in which the substances are mixed. The assay involves measuring the time of flow of the droplet at a predetermined distance along the microfluidic channel. The time of flow of the droplet, for which the agglutination reaction occurred, is significantly longer than the analogous time for droplet for which no reaction occurred; the comparison of the times of flow allows to distinguish whether the agglutination occurred or not.
Thus, there exists a need for further development of measurement of the changes in physical properties of liquids when flowing through microfluidic channels. Various methods have been described to address this need. For example, the following methods have been proposed for measuring of additional resistance of droplets in microfluidic channels:
V. Labrot et al., Biomicrofluidics, vol. 3, p. 012804, 2009, describes a method for direct measuring of the pressure droplet on a short section of a channel during the flow of a single droplet. Nonetheless, it requires the use of a very precise micromanometer and causes additional errors due to the presence of side channels for measuring the pressure.
Another method, described in S. A. Vanapalli et al., Lab on a Chip, vol. 9, p. 982, 2009, features a flow comparator based on balancing the measured flow (with droplet) with a dyed reference flow of controlled rate. Nonetheless, the quality of this method is limited by the experimental indication of the displacement of interface between dyed and clear oil.
Yet another known method, described in M. J. Fuerstman et al., Lab on a Chip, vol. 7, p. 1479, 2007 and V. Labrot et al., Biomicrofluidics, vol. 3, p. 012804, 2009 utilizes the model of flow of discrete segments of fluids through a simple loop of channels by measuring the velocity of flow of bubbles or droplets. The accuracy of this method depends critically on a number of assumptions and technical details: the behavior of droplets at diverging junctions and the spatial resolution of the measurements.
The methods quoted above are based on observations and measurements of properties of individual droplets.
It follows from the above-mentioned publications that the techniques of measurement of the change in certain physical properties of liquid samples with the microfluidic channels undergo fast development.
There exists a need for further development of agglutination test methods based on measurement of change in physical properties of a liquid sample, which will be more efficient and will lead to more reliable results, even whilst providing reduced sample size, such as droplets.